Sorescu D, Somers M J, Lassègue B, Grant S, Harrison D G, Griendling K K
Department of Medicine, Division of Cardiology, Emory University, Atlanta, GA 30322, USA.
Free Radic Biol Med. 2001 Mar 15;30(6):603-12. doi: 10.1016/s0891-5849(00)00507-4.
Endogenously produced reactive oxygen species are important for intracellular signaling mechanisms leading to vascular smooth muscle cell (VSMC) growth. It is therefore critical to define the potential enzymatic sources of ROS and their regulation by agonists in VSMCs. Previous studies have investigated O2*- production using lucigenin-enhanced chemiluminescence. However, lucigenin has been recently criticized for its ability to redox cycle and its propensity to measure cellular reductase activity independent from O2*-. To perform a definitive characterization of VSMC oxidase activity, we used electron spin resonance trapping of O2*- with DEPMPO. We confirmed that the main source of O2*- from VSMC membranes is an NAD(P)H oxidase and that the O2*- formation from mitochondria, xanthine oxidase, arachidonate-derived enzymes, and nitric oxide synthases in VSMC membranes was minor. The VSMC NAD(P)H oxidase(s) are able to produce more O2*- when NADPH is used as the substrate compared to NADH (the maximal NADPH signal is 2.4- +/- 0.4-fold higher than the NADH signal). The two substrates had similar EC(50)'s ( approximately 10-50 microM). Stimulation with angiotensin II and platelet-derived growth factor also predominantly increased the NADPH-driven signal (101 +/- 8% and 83 +/- 1% increase above control, respectively), with less of an effect on NADH-dependent O2*- (17 +/- 3% and 36 +/- 5% increase, respectively). Moreover, incubation of the cells with diphenylene iodonium inhibited predominantly NADPH-stimulated O2*-. In conclusion, electron spin resonance characterization of VSMC oxidase activity supports a major role for an NAD(P)H oxidase in O2*- production in VSMCs, and provides new evidence concerning the substrate dependency and agonist-stimulated activity of this key enzyme.
内源性产生的活性氧对于导致血管平滑肌细胞（VSMC）生长的细胞内信号传导机制很重要。因此，确定VSMC中ROS的潜在酶源及其受激动剂的调节至关重要。先前的研究使用光泽精增强化学发光法研究了超氧阴离子（O2*-）的产生。然而，光泽精最近因其氧化还原循环能力以及独立于O2*-测量细胞还原酶活性的倾向而受到批评。为了对VSMC氧化酶活性进行明确的表征，我们使用DEPMPO对O2*-进行电子自旋共振捕获。我们证实，VSMC膜中O2*-的主要来源是NAD(P)H氧化酶，而VSMC膜中线粒体、黄嘌呤氧化酶、花生四烯酸衍生酶和一氧化氮合酶产生的O2*-较少。与NADH相比，当使用NADPH作为底物时，VSMC的NAD(P)H氧化酶能够产生更多的O2*-（最大NADPH信号比NADH信号高2.4±0.4倍）。两种底物具有相似的半数有效浓度（EC(50)）（约10-50μM）。用血管紧张素II和血小板衍生生长因子刺激也主要增加了NADPH驱动的信号（分别比对照增加101±8%和83±1%），对NADH依赖性O2*-的影响较小（分别增加17±3%和36±5%）。此外，用二苯基碘鎓孵育细胞主要抑制NADPH刺激的O2*-。总之，VSMC氧化酶活性的电子自旋共振表征支持NAD(P)H氧化酶在VSMC中O2*-产生中的主要作用，并提供了关于这种关键酶的底物依赖性和激动剂刺激活性的新证据。
